Testosterone Differentially Affects T Cells and Neurons in Murine and Human Models of Neuroinflammation and Neurodegeneration

The high female-to-male sex ratio of multiple sclerosis (MS) prevalence has continuously confounded researchers, especially in light of male patients' accelerated disease course at later stages of MS. Although multiple studies have concentrated on estrogenic mechanisms of disease modulation, fairly little attention has been paid to androgenic effects in a female system, and even fewer studies have attempted to dissociate hormonal effects on the neurodegenerative and neuroinflammatory processes of MS. Herein, we demonstrate the differential effects of hormone treatment on the acute inflammatory and chronic neurodegenerative phases of murine experimental autoimmune encephalomyelitis. Although s.c. treatment with testosterone and aromatase inhibitor applied beginning on the day of immunization ameliorated initial course of disease, similar treatment administered therapeutically exacerbated chronic disease course. Spinal cord analyses of axonal densities reflected the clinical scores of the chronic phase. In vitro, testosterone treatment not only decreased Th1 and Th17 differentiation in an aromatase-independent fashion, but also exacerbated cell death in induced pluripotent stem cell–derived primary human neurons under oxidative stress conditions in an aromatase inhibitor–dependent manner. Thus, through the alleviation of inflammatory processes and the exacerbation of neurodegenerative processes, androgens may contribute to the epidemiologic sex differentials observed in MS prevalence and course. The high female-to-male sex ratio of multiple sclerosis (MS) prevalence has continuously confounded researchers, especially in light of male patients' accelerated disease course at later stages of MS. Although multiple studies have concentrated on estrogenic mechanisms of disease modulation, fairly little attention has been paid to androgenic effects in a female system, and even fewer studies have attempted to dissociate hormonal effects on the neurodegenerative and neuroinflammatory processes of MS. Herein, we demonstrate the differential effects of hormone treatment on the acute inflammatory and chronic neurodegenerative phases of murine experimental autoimmune encephalomyelitis. Although s.c. treatment with testosterone and aromatase inhibitor applied beginning on the day of immunization ameliorated initial course of disease, similar treatment administered therapeutically exacerbated chronic disease course. Spinal cord analyses of axonal densities reflected the clinical scores of the chronic phase. In vitro, testosterone treatment not only decreased Th1 and Th17 differentiation in an aromatase-independent fashion, but also exacerbated cell death in induced pluripotent stem cell–derived primary human neurons under oxidative stress conditions in an aromatase inhibitor–dependent manner. Thus, through the alleviation of inflammatory processes and the exacerbation of neurodegenerative processes, androgens may contribute to the epidemiologic sex differentials observed in MS prevalence and course. The female-to-male prevalence ratio of multiple sclerosis (MS) presently stands at 3:1, if not more; however, male MS patients tend to have not only a slightly later onset but also a more debilitating disease course than do their female counterparts, especially during the later stages of MS progression.1Bove R. Chitnis T. Sexual disparities in the incidence and course of MS.Clin Immunol. 2013; 149: 201-210Crossref PubMed Scopus (73) Google Scholar Although environmental and genetic factors have been explored,2Chao M.J. Herrera B.M. Ramagopalan S.V. Deluca G. Handunetthi L. Orton S.M. Lincoln M.R. Sadovnick A.D. Ebers G.C. Parent-of-origin effects at the major histocompatibility complex in multiple sclerosis.Hum Mol Genet. 2010; 19: 3679-3689Crossref PubMed Scopus (37) Google Scholar, 3Du S. Itoh N. Askarinam S. Hill H. Arnold A.P. Voskuhl R.R. XY sex chromosome complement, compared with XX, in the CNS confers greater neurodegeneration during experimental autoimmune encephalomyelitis.Proc Natl Acad Sci U S A. 2014; 111: 2806-2811Crossref PubMed Scopus (88) Google Scholar, 4Costenbader K.H. Gay S. Alarcón-Riquelme M.E. Iaccarino L. Doria A. Genes, epigenetic regulation and environmental factors: which is the most relevant in developing autoimmune diseases?.Autoimmun Rev. 2012; 11: 604-609Crossref PubMed Scopus (168) Google Scholar much research attempting to elucidate this topic has been dedicated to sex steroid hormones. Although epidemiological and linkage studies have suggested a role of hormones in the disease,5Voskuhl R.R. Gold S.M. Sex-related factors in multiple sclerosis susceptibility and progression.Nat Rev Neurol. 2012; 8: 255-263Crossref PubMed Scopus (179) Google Scholar, 6Pakpoor J. Goldacre R. Schmierer K. Giovannoni G. Goldacre M.J. Testicular hypofunction and multiple sclerosis risk: a record-linkage study.Ann Neurol. 2014; 76: 625-628Crossref PubMed Scopus (29) Google Scholar, 7Bove R. Musallam A. Healy B.C. Raghavan K. Glanz B.I. Bakshi R. Weiner H. De Jager P.L. Miller K.K. Chitnis T. Low testosterone is associated with disability in men with multiple sclerosis.Mult Scler. 2014; 20: 1584-1592Crossref PubMed Scopus (78) Google Scholar clinical trials in humans have provided further evidence of a direct influence of sex steroid hormones on the pathogenesis and disease course of MS.8Sicotte N.L. Liva S.M. Klutch R. Pfeiffer P. Bouvier S. Odesa S. Wu T.C. Voskuhl R.R. Treatment of multiple sclerosis with the pregnancy hormone estriol.Ann Neurol. 2002; 52: 421-428Crossref PubMed Scopus (377) Google Scholar, 9Voskuhl R.R. Wang H. Wu T.C.J. Sicotte N.L. Nakamura K. Kurth F. Itoh N. Bardens J. Bernard J.T. Corboy J.R. Cross A.H. Dhib-Jalbut S. Ford C.C. Frohman E.M. Giesser B. Jacobs D. Kasper L.H. Lynch S. Parry G. Racke M.K. Reder A.T. Rose J. Wingerchuk D.M. MacKenzie-Graham A.J. Arnold D.L. Tseng C.H. Elashoff R. Estriol combined with glatiramer acetate for women with relapsing-remitting multiple sclerosis: a randomised, placebo-controlled, phase 2 trial.Lancet Neurol. 2016; 15: 35-46Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 10Huang G. Wharton W. Travison T.G. Ho M.H. Gleason C. Asthana S. Bhasin S. Basaria S. Effects of testosterone administration on cognitive function in hysterectomized women with low testosterone levels: a dose-response randomized trial.J Endocrinol Invest. 2014; 38: 455-461Crossref PubMed Scopus (16) Google Scholar, 11Kurth F. Luders E. Sicotte N.L. Gaser C. Giesser B.S. Swerdloff R.S. Montag M.J. Voskuhl R.R. Mackenzie-Graham A. Neuroprotective effects of testosterone treatment in men with multiple sclerosis.Neuroimage Clin. 2014; 4: 454-460Crossref PubMed Scopus (81) Google Scholar, 12Sicotte N.L. Giesser B.S. Tandon V. Klutch R. Steiner B. Drain A.E. Shattuck D.W. Hull L. Wang H.-J. Elashoff R.M. Testosterone treatment in multiple sclerosis: a pilot study.Arch Neurol. 2007; 64: 683-688Crossref PubMed Scopus (161) Google Scholar Within these studies, the different effects of steroid hormone treatment on cognitive ability and gadolinium-enhancing lesions indicate a plausible dissociation between hormonal effects on the neurodegenerative and neuroinflammatory processes of MS. This conjecture is strengthened by findings from in vitro experiments demonstrating that the timing of hormone treatment to cellular stressor,13Holmes S. Abbassi B. Su C. Singh M. Cunningham R.L. Oxidative stress defines the neuroprotective or neurotoxic properties of androgens in immortalized female rat dopaminergic neuronal cells.Endocrinology. 2013; 154: 4281-4292Crossref PubMed Scopus (53) Google Scholar strength of hormone treatment,14Orlando R. Caruso A. Molinaro G. Motolese M. Matrisciano F. Togna G. Melchiorri D. Nicoletti F. Bruno V. Nanomolar concentrations of anabolic–androgenic steroids amplify excitotoxic neuronal death in mixed mouse cortical cultures.Brain Res. 2007; 1165: 21-29Crossref PubMed Scopus (53) Google Scholar type of cellular stressor,15Nguyen T.V. Jayaraman A. Quaglino A. Pike C.J. Androgens selectively protect against apoptosis in hippocampal neurones.J Neuroendocrinol. 2010; 22: 1013-1022Crossref PubMed Scopus (55) Google Scholar and strength of stressor13Holmes S. Abbassi B. Su C. Singh M. Cunningham R.L. Oxidative stress defines the neuroprotective or neurotoxic properties of androgens in immortalized female rat dopaminergic neuronal cells.Endocrinology. 2013; 154: 4281-4292Crossref PubMed Scopus (53) Google Scholar can result in differing cellular outputs. Indeed, such may provide a partial explanation of why female MS patients typically experience greater inflammatory lesions, whereas their male counterparts display more neurodegeneration.5Voskuhl R.R. Gold S.M. Sex-related factors in multiple sclerosis susceptibility and progression.Nat Rev Neurol. 2012; 8: 255-263Crossref PubMed Scopus (179) Google Scholar This question of differential hormonal actions has yet to be fully explored within the context of MS. Most animal studies have treated the arguably biphasic disease as a single entity,16Palaszynski K.M. Liu H. Loo K.K. Voskuhl R.R. Estriol treatment ameliorates disease in males with experimental autoimmune encephalomyelitis: implications for multiple sclerosis.J Neuroimmunol. 2004; 149: 84-89Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 17MacKenzie-Graham A.J. Rinek G.A. Avedisian A. Morales L.B. Umeda E. Boulat B. Jacobs R.E. Toga A.W. Voskuhl R.R. Estrogen treatment prevents gray matter atrophy in experimental autoimmune encephalomyelitis.J Neurosci Res. 2012; 90: 1310-1323Crossref PubMed Scopus (22) Google Scholar, 18Morales L.B.J. Loo K.K. Liu H. Peterson C. Tiwari-Woodruff S. Voskuhl R.R. Treatment with an estrogen receptor α ligand is neuroprotective in experimental autoimmune encephalomyelitis.J Neurosci. 2006; 26: 6823-6833Crossref PubMed Scopus (136) Google Scholar and those that have teased apart the aforementioned processes have mostly focused on the beneficial estrogenic effects on immune system and myelin pathology.19Haghmorad D. Amini A.A. Mahmoudi M.B. Rastin M. Hosseini M. Mahmoudi M. Pregnancy level of estrogen attenuates experimental autoimmune encephalomyelitis in both ovariectomized and pregnant C57BL/6 mice through expansion of Treg and Th2 cells.J Neuroimmunol. 2014; 277: 85-95Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 20Wisdom A.J. Cao Y. Itoh N. Spence R.D. Voskuhl R.R. Estrogen receptor-β ligand treatment after disease onset is neuroprotective in the multiple sclerosis model.J Neurosci Res. 2013; 91: 901-908Crossref PubMed Scopus (27) Google Scholar, 21Wu W. Tan X. Dai Y. Krishnan V. Warner M. Gustafsson J.-Å. Targeting estrogen receptor β in microglia and T cells to treat experimental autoimmune encephalomyelitis.Proc Natl Acad Sci U S A. 2013; 110: 3543-3548Crossref PubMed Scopus (111) Google Scholar Similarly, although in vitro experiments have mostly confirmed positive estrogenic (typically via estradiol) effects on neuron survival22Bains M. Cousins J.C. Roberts J.L. Neuroprotection by estrogen against MPP+-induced dopamine neuron death is mediated by ERα in primary cultures of mouse mesencephalon.Exp Neurol. 2007; 204: 767-776Crossref PubMed Scopus (46) Google Scholar, 23Park S.Y. Tournell C. Sinjoanu R.C. Ferreira A. Caspase-3- and calpain-mediated tau cleavage are differentially prevented by estrogen and testosterone in beta-amyloid-treated hippocampal neurons.Neuroscience. 2007; 144: 119-127Crossref PubMed Scopus (84) Google Scholar and inflammation,24Lélu K. Laffont S. Delpy L. Paulet P.-E. Périnat T. Tschanz S.A. Pelletier L. Engelhardt B. Guéry J.-C. Estrogen receptor α signaling in T lymphocytes is required for estradiol-mediated inhibition of Th1 and Th17 cell differentiation and protection against experimental autoimmune encephalomyelitis.J Immunol. 2011; 187: 2386-2393Crossref PubMed Scopus (159) Google Scholar, 25Saijo K. Collier J.G. Li A.C. Katzenellenbogen J.A. Glass C.K. An ADIOL-ERβ-CtBP transrepression pathway negatively regulates microglia-mediated inflammation.Cell. 2011; 145: 584-595Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar the few investigations into androgenic effects have achieved less consensus. Though most in vitro inflammation research has demonstrated convincing evidence of pure androgenic protection via the induction of anti-inflammatory cell profiles,26Liva S.M. Voskuhl R.R. Testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production.J Immunol. 2001; 167: 2060-2067Crossref PubMed Scopus (289) Google Scholar, 27Fijak M. Schneider E. Klug J. Bhushan S. Hackstein H. Schuler G. Wygrecka M. Gromoll J. Meinhardt A. Testosterone replacement effectively inhibits the development of experimental autoimmune orchitis in rats: evidence for a direct role of testosterone on regulatory T cell expansion.J Immunol. 2011; 186: 5162-5172Crossref PubMed Scopus (142) Google Scholar, 28Vignozzi L. Cellai I. Santi R. Lombardelli L. Morelli A. Comeglio P. Filippi S. Logiodice F. Carini M. Nesi G. Gacci M. Piccinni M.-P. Adorini L. Maggi M. Antiinflammatory effect of androgen receptor activation in human benign prostatic hyperplasia cells.J Endocrinol. 2012; 214: 31-43Crossref PubMed Scopus (73) Google Scholar congruent studies on neurodegeneration and protection have resulted in conflicting results29Estrada M. Varshney A. Ehrlich B.E. Elevated testosterone induces apoptosis in neuronal cells.J Biol Chem. 2006; 281: 25492-25501Crossref PubMed Scopus (120) Google Scholar, 30Hammond J. Le Q. Goodyer C. Gelfand M. Trifiro M. LeBlanc A. Testosterone-mediated neuroprotection through the androgen receptor in human primary neurons.J Neurochem. 2001; 77: 1319-1326Crossref PubMed Scopus (265) Google Scholar, 31Nguyen T.V. Yao M. Pike C.J. Androgens activate mitogen-activated protein kinase signaling: role in neuroprotection.J Neurochem. 2005; 94: 1639-1651Crossref PubMed Scopus (143) Google Scholar or findings have been confounded by the potential conversion of testosterone to estradiol within a given system.13Holmes S. Abbassi B. Su C. Singh M. Cunningham R.L. Oxidative stress defines the neuroprotective or neurotoxic properties of androgens in immortalized female rat dopaminergic neuronal cells.Endocrinology. 2013; 154: 4281-4292Crossref PubMed Scopus (53) Google Scholar Thus, to uncover the contribution of androgens to MS and experimental autoimmune encephalomyelitis (EAE) disease course, experiments conducted in vivo must dissociate neurodegeneration from inflammatory processes, whereas those in vitro ought to use disease-appropriate paradigms. Here, we successfully dissociate these two aforementioned processes in vivo and investigate androgen specificity on these with the coapplication of testosterone and the aromatase inhibitor fadrozole to prevent endogenous estradiol conversion. Strategically-timed hormonal treatments targeting the acute (inflammatory) or chronic (neurodegenerative) phases of the C57Bl/6 murine, myelin oligodendrocyte protein (Mog)-induced MS model EAE20Wisdom A.J. Cao Y. Itoh N. Spence R.D. Voskuhl R.R. Estrogen receptor-β ligand treatment after disease onset is neuroprotective in the multiple sclerosis model.J Neurosci Res. 2013; 91: 901-908Crossref PubMed Scopus (27) Google Scholar revealed an androgen-mediated alleviation of inflammation and exacerbation of neurodegeneration. In vitro murine T-cell differentiations along with oxidative stress–induced neurodegeneration assays in human primary neuron cultures derived from induced pluripotent stem cells have confirmed these differential androgenic effects. Herein, we describe how androgens differentially affect the inflammatory and neurodegenerative processes in the context of EAE and in vitro models. Gonadally intact female C57BL/6J mice, aged 7.5 to 10 weeks, were obtained from Charles River (Sulzfeld, Germany) or Janvier (Saint-Berthevin, France) and maintained under a 12:12-hour light–dark schedule. All experiments were conducted with the approval of the Regulatory Authority of the Federal State North-Rhine Westfalia handling all cases dealing with environment and consumer protection (study reference number 84-02.04.2015.A259). Mice were immunized by two 5-μL bilateral s.c. flank injections containing 50 μg Mog 35-55 peptide (Charité, Berlin, Germany) and 50 μg complete Freund's adjuvant (Difco Laboratories, Detroit, MI) in a 1:1 solution of phosphate-buffered saline (PBS) and incomplete Freund's adjuvant (BD, Heidelberg, Germany). Immediately after and 2 days after immunization, mice received 100 μL i.p. PBS injections containing 0.2 μg pertussis toxin (Millipore, Darmstadt, Germany). Mice were actively immunized as described in Active EAE Induction, though total Mog and complete Freund's adjuvant injected were 400 μg and 100 μg, respectively. After daily hormone treatments (Hormone Treatments), spleen and lymph nodes were isolated on the 9th day after immunization (D9) and cells cultivated in restimulation medium (RPMI supplemented with 1% pen/strep, 1% nonessential amino acids, 100 mmol/L sodium pyruvate, 200 mmol/L l-glutamine, 10% fetal calf serum, and 50 μmol/L β-mercaptoethanol). Cells were first stimulated with 50 μg/mL Mog, 25 ng/mL IL-12, and 10 μg/mL α-IL-4, and after 24 hours 5 ng/mL IL-2 was added to the medium. On the 3rd day after isolation, 10 million cells in 500 μL were i.p. injected into 15 female mice (8 weeks old; Janvier). Two animals per treatment group were sacrificed on D14 for CD3 and macrophage differentiation antigen (Mac3) quantification (IHC Analysis). The remaining animals were immunized with Mog and complete Freund's adjuvant as in the active immunization beginning on D13. Animals were weighed and scored daily based on a 10-point EAE scale: 0 indicates no signs of disease; 1 indicates reduced tail tone; 2 indicates entire tail tone lost; 3 indicates rolling gait and mild ataxia; 4 indicates rolling gait, moderate ataxia, and/or mild paresis of hind limbs; 5 indicates severe ataxia and paraparesis of hind limbs; 6 indicates severe paresis of both or complete paralysis of one hind limb; 7 indicates complete hind limb paralysis; 8 indicates forelimb weakness or tetraplegia; 9 indicates respiratory distress, moribund; and 10 indicates death. Testosterone (T) and the aromatase inhibitor fadrozole (FAD) were both obtained from Sigma-Aldrich (Hamburg, Germany). Stock solutions of 46.2 mmol/L T and 77.0 mmol/L FAD were prepared in 100% ethanol and stored at −20°C. Working solutions prepared in Miglyol 812 N oil (generously provided by Cremer Oleo, Witten, Germany) contained 10% ethanol and, where indicated, 4.16 mmol/L T and/or 0.77 mmol/L FAD. These concentrations yielded treatments of 60 μg and 10 μg, respectively, as derived from previous studies.26Liva S.M. Voskuhl R.R. Testosterone acts directly on CD4+ T lymphocytes to increase IL-10 production.J Immunol. 2001; 167: 2060-2067Crossref PubMed Scopus (289) Google Scholar, 32McCullough L.D. Blizzard K. Simpson E.R. Oz O.K. Hurn P.D. Aromatase cytochrome P450 and extragonadal estrogen play a role in ischemic neuroprotection.J Neurosci. 2003; 23: 8701-8705Crossref PubMed Google Scholar Vehicle controls contained 10% ethanol in either PBS (treatment in acute phase) or Miglyol 812 N oil (treatment in chronic phase). Injections of working solutions s.c. into the cervical area were accomplished using 23-gauge needles. To reduce inconstant hormone fluctuations, injections were administered within the same 3-hour window each day. Animals were randomized into four groups (T, T + FAD, FAD, and vehicle) containing similar mean weights and housed together before EAE induction. Beginning on the day of immunization (day 0), mice were injected daily with hormone working solutions or PBS control. PBS was used as control due to the possibility of isolated oil treatment (composed of short-chain fatty acids) affecting inflammation33Haghikia A. Jörg S. Duscha A. Berg J. Manzel A. Waschbisch A. Hammer A. Lee D.-H. May C. Wilck N. Balogh A. Ostermann A.I. Schebb N.H. Akkad D.A. Grohme D.A. Kleinewietfeld M. Kempa S. Thöne J. Demir S. Müller D.N. Gold R. Linker R.A. Dietary fatty acids directly impact central nervous system autoimmunity via the small intestine.Immunity. 2015; 43: 817-829Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar; hormone treatment with oil overpowered this effect in treated animals. For active acute EAE, treatment was continued daily until sacrifice (days 27 to 30); for adoptive transfer, hormone treatments were given only to donor mice until sacrifice for transfer. Animals were randomly, individually treated at the first sign of disease onset (days 8 to 14 after immunization) with either hormone injections (T or T + FAD) or Miglyol-based vehicle as control. Treatment was continued daily until sacrifice (days 43 to 44). At experiment termination, mice were deeply anesthetized with a narcotics solution containing 0.0375% Ventraquil (Ceva Sante Animale, Libourne, France), 1.8 mg/mL Xylavet (CP-Pharma, Burgdorf, Germany), and 37.5 mg/mL ketamine (CP-Pharma) in 0.9% sodium chloride. Once reflexes became dormant, a transcardial perfusion was performed with 0.9% sodium chloride followed by 4% paraformaldehyde solution. Spinal cords, brain (excluding olfactory bulbs), and spleen were removed and submerged in 4% paraformaldehyde for 24 to 36 hours before 24 to 48 hours cryoprotection in PBS. Cassettes containing three sections of cervical, thoracic, and lumbar spine sections, as well as cassettes prepared with quartered brain slices, were subjected to serial dehydration using the TP1020 Semienclosed Benchtop Tissue Processor (Leica, Wetzlar, Germany). Tissue was subjected to ten 1-hour reagent baths (50, 70, 80, 90, 95, 3 × 100% ethanol, and 2× Xylol) before paraffin wax bath. Cassettes were subsequently embedded in paraffin blocks using the EG1160 Embedding Center (Leica). Free-floating sections (5 μm thick) were sliced with a RM2155 Microtome (Leica) and mounted onto SuperFrost Plus slides (Gerhard Menzel, Braunschweig, Germany) in preparation for staining and analyses. Previously dehydrated, paraffin-embedded tissue was sectioned into 5-μm slices and subjected to rehydration via a descending alcohol series (beginning with xylene and followed by 99%, 90%, 80%, 70%, and 50% ethanol submersions) before Bielschowsky silver impregnation, Luxol fast blue staining, or diaminobenzidine staining for CD3+ T cells and Mac3+ macrophages. Axonal damage in spinal cord white matter was evaluated via silver staining procedures. Briefly, after sections were incubated with 10% silver nitrate for 20 minutes, cells were stored in distilled water (dH2O); 25% ammonia solution was added until solution clarity. Tissue sections were then incubated in the clear silver hydroxide solution for another 20 minutes. Slides were washed in 1% ammonia solution before being subjected to constant shaking and the addition of developer solution (4.17 mg/mL citric acid, 1.665% formaldehyde, and one drop 65% HNO3 in 120 mL dH2O). After final wash steps in 1% ammonia and dH2O, slides were incubated in 5% sodium thiosulfate for 3 minutes before ascending alcohol series and coverslipping. Luxol fast blue staining was used for observing demyelination within spinal cord white matter. Slides were initially incubated in Luxol fast blue staining solution overnight at 60°C. Slides were then rinsed in 96% ethanol and dH2O before 0.05% lithium carbonate exposure. After this, sections were washed in dH2O and 70% ethanol before a 10-minute incubation with 0.8% periodic acid. Slides were subsequently counterstained with Schiff reagent for 40 minutes. Final washing steps with sulfite and flowing water and an ascending alcohol series preceded coverslipping. Spinal cord infiltrates, namely CD3+ T cells and Mac3+ macrophages, were visualized using diaminobenzidine staining. After antigen unmasking via a 35-minute bath in heated citrate buffer, sections were blocked with a 10% bovine serum albumin solution in PBS for 30 minutes. Slides were then incubated in the primary antibody solutions (1:200 in 1% bovine serum albumin in PBS) of either anti-CD3 (Serotec, Düsseldorf, Germany) or anti-Mac3 (BD Biosciences, Heidelberg, Germany) overnight at 4°C. This incubation was followed by a 15-minute bath in 3% hydrogen peroxide to block endogenous peroxidase activity. Incubation with the secondary, biotinylated antibody (biotinylated rabbit anti-rat, Vector Laboratories, Dossenheim, Germany; 1:200 dilution in 1% bovine serum albumin in PBS) was performed over 1 hour, followed by incubation with an avidin–biotin complex (Vectastain ABC Kit; Vector) according to the manufacturer's instructions. Tissue was then transferred into diaminobenzidine staining solution for 10 minutes and counterstained with hematoxylin at a 1:1 dilution in dH2O for 20 seconds. Sections were washed in flowing water before ascending alcohol series exposure and coverslipping. To quantify the impact of androgens on spinal cord axonal preservation, demyelination, and T-cell/macrophage infiltration, nine independent spinal cord cross sections per specimen were analyzed. Demyelination was quantified semiautomatically using CellD software version 2.6 (Olympus, Hamburg, Germany), and was defined as the ratio to total white matter area. Axonal densities were determined by axon counting of profiles in three lesions per section on a 100-mm-diameter grid at 1000× magnification. Cellular infiltrates were counted in three visual fields within each of the cervical, thoracic, and lumbar sections as captured at 20× magnification (image size 264.6 × 356.6 μm) and multiplied by a factor of 10.6 to determine the number of cells per square millimeter. Splenic T cells were isolated with magnetic activated cell sorting via the Pan T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions, using 30 million cells. For naïve differentiations, cells were fluorescently stained for 30 minutes in an antibody cocktail containing αCD4-fluorescein isothiocyanate (eBioscience, Frankfurt, Germany), αCD44-phycoerythrin (BioLegend, London, UK), αCD62L–antigen-presenting cells (eBioscience), and αCD25-phycoerythrin-Cy5 (eBioscience). Naïve cells were subsequently isolated by fluorescence-activated cell sorting with MoFlo (Beckman Coulter, Krefeld, Germany) and the fluorescence-activated cell sorting–core in the department of neurology, in the neuroimmunological laboratory of the University of Erlangen. For Th17 differentiation, sorted naïve T cells (CD4+CD26L+CD44lowCD25–) were stimulated by 2 μg/mL plate-bound anti-CD3 (BD Biosciences) and 2 μg/mL anti-CD28 (BD Biosciences) in the presence of 40 ng/mL IL-6 and 1 ng/mL recombinant human transforming growth factor β1 for 4 days. For Th1 differentiations, sorted naïve CD4+ T cells were incubated at 37°C, 5% CO2 for 96 hours with anti-CD3, anti-CD28, 20 ng/mL IL-12, and 10 mg/mL anti-IL-4 (BioLegend). To determine the influence of testosterone on T-cell differentiation, three independent platings of cells were cultured with and without 1, 10, and 100 nmol/L T. To ensure the observed hormonal effects were androgenic in nature, 5 μmol/L FAD34Chronowska E. Tománek M. Kott T. Effect of aromatase inhibitor (fadrozole) on proliferation, estradiol production and telomerase activity in pig granulosa cells in vitro.Czech J Anim Sci. 2009; 54: 566-574Google Scholar was simultaneously applied in one experiment. For intracellular flow cytometry, cells were stimulated for 4 hours with 1 μmol/L ionomycin and 50 ng/mL phorbol-myristate-acetate in the presence of 2 μmol/L monesin and stained for CD4 (eBioscience), intracellular IL-17A (eBioscience), and interferon-γ (eBioscience). The fixable viability dye eFluor780 (0.2 μL/test; Thermo Fisher Scientific, Darmstadt, Germany) was used for excluding dead cells. Primary human neuron cultures were cultured from renal cells found in female healthy control donor urine via an induced pluripotent stem cell procedure as previously described.35Massa M.G. Gisevius B. Hirschberg S. Hinz L. Schmidt M. Gold R. Prochnow N. Haghikia A. Multiple sclerosis patient-specific primary neurons differentiated from urinary renal epithelial cells via induced pluripotent stem cells.PLoS One. 2016; 11 (Kleinschnitz C, editor): e0155274Crossref Scopus (12) Google Scholar Previously frozen cells were plated directly onto poly-l-ornithine hydrobromide/laminin-coated 96-well plates (5000 to 10,000 cells per well) at least 3 days before testing. By testing date, all neurons were at least 20 days old. Oxidative stress (15 μmol/L H2O2) was applied for 4 to 5 hours before the application of hormone treatment (10, 100, or 1000 nmol/L T with or without 5 μmol/L FAD, 0.015% ethanol). After 24 hours of H2O2 exposure and 19 to 20 hours of hormone treatment, cells were washed once before the application of 4 μg/mL Hoechst solution. After a 2-hour incubation, 0.5 μL 7-aminoactinomycin D viability stain per well was added for 10 minutes before fluorescent microscopy. Because Hoechst integrates into cell DNA regardless of cell status, whereas 7-aminoactinomycin D binds DNA only on membrane fractionation, apoptotic cells are double-stained. Cells were analyzed via cell counting at 20× magnification, with three visual fields pooled from each well. All conditions had n ≥ 3 with triplicates. Data are presented as percent double-stained of total Hoechst-stained nuclei. All statistical analyses were performed with Prism software version 6 (GraphPad Software, La Jolla, CA). Three individual EAE experiments were performed, and data from the same condition were pooled for clinical scoring and immunohistochemistry analysis. EAE score data were analyzed via Kruskal-Wallis test followed by Dunn multiple comparisons test;